The overall objective of the proposal is to develop a novel protein expression platform by capitalizing on the remarkable properties of the recently discovered twin-arginine translocation (Tat) pathway of bacteria. With over 300 therapeutic proteins currently in various stages of clinical trials, the road to a healthier future will require new methods for producing safer and less expensive recombinant proteins. For this purpose, the recently discovered twin-arginine translocation (Tat) pathway will be utilized as a novel recombinant protein expression platform in Escherichia coli. Towards this objective, Phase I encompasses the following specific aims: 1) to define the secretory capacity of the bacterial Tat transporter; 2) to optimize Tat secretion efficiency via chaperone co-expression strategies; and 3) to scale-up the Tat-based protein production process. This research is expected to not only result in the development a novel platform for bacterial protein expression but will also facilitate a deeper understanding of a poorly understood biological mechanism. Phase II will entail a much broader effort to study and improve Tat transport efficiency including, for instance, directed evolution strategies to engineer "hyperactive" Tat transporters, cell engineering methods to optimize the host for Tat expression and leader peptide optimization studies. In addition, we expect to fully develop scale-up methods for large-scale Tat expression by using optimized carbon-source feed strategies, as well as temperature, dissolved oxygen and pH optimization.